The proposed study represents a continuation of our efforts to understand mechanisms of segmentation and homeotic gene activity in the early Drosophila embryo. Particular efforts will focus on the visualization of noncoding RNAs using RNA FISH and confocal microscopy, as well as the function and regulation of specific microRNAs. Transvection is a form of genetic complementation, whereby mutations in a gene can be complemented in trans by the other copy of the gene on the other homologue. We have recently employed a newly developed RNA FISH method to visualize trans-homologue enhancer-promoter interactions in the early embryo. The analysis of the Abd-B Hox locus suggests that such interactions are common: something like 25% of all transcripts appear to arise from trans-homologue interactions. The proposed research plan includes a number of experiments to examine transvection at other Hox loci and determine the underlying mechanism. One of the major mechanisms governing localized patterns of Hox gene expression is posterior prevalence, whereby posterior Hox proteins repress the transcription of more anterior Hox genes. However, it has been recently shown that the intergenic regions of Hox complexes encode noncoding RNAs that are processed into microRNAs. We will examine the possibility that these miRNAs inhibit the expression of anterior Hox proteins. If so, this would be consistent with early genetic studies suggesting that the Bithorax complex contains at least 8 genes, even though only 3 Hox transcription units are known. The final goal of the proposed study is to investigate how Bicoid, along with just a few gap genes, produce so many different stripes of pair-rule gene expression in the early embryo. We will investigate the possibility that gap repressers recruit different corepressor proteins when bound to distinct stripe enhancers. In addition, whole-genome tiling arrays will be used to determine whether noncoding target genes of the Bicoid gradient are important for segmentation.